Extraction of high-quality bacterial RNA from infected leaf tissue for bacterial in planta gene expression analysis by multiplexed fluorescent Northern hybridization.

Molecular Plant Pathology
Alexander SchenkMatthias S Ullrich

Abstract

Plant pathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. In planta gene expression analysis is important to understand the impact of these genes on bacterial virulence. A new mRNA-based approach using multiplexed Northern hybridization was developed. High-quality bacterial and plant total RNA was successfully isolated from leaf tissue infiltrated with Pseudomonas syringae. The procedure employs a new extraction buffer formulation containing glycine, sodium dodecylsulphate, cetyltrimethylammonium bromide, high-molecular-weight polyethylene glycol and beta-mercaptoethanol. Cell lysis and classical acid-phenol extraction steps followed by LiCl precipitation yielded large amounts of total RNA of high purity and integrity. Multiplexing of DIG and chemically fluorescently labelled RNA probes was developed and expression data were normalized using the 23S rRNA gene as reference. The method was validated by studying in planta expression of the P. syringae genes mucD, cmaA, cfl, corR, corS and corP comprising a selection of highly expressed biosynthetic and low-expressed regulatory genes. The method was assessed regarding its sensitivity and might by useful for studying a variety of ...Continue Reading

References

Dec 1, 2001·Molecular Biotechnology·L JaakolaA Hohtola
Nov 20, 2002·Molecular Plant-microbe Interactions : MPMI·Bart P J GeraatsL C van Loon
Oct 23, 2004·Molecular Plant-microbe Interactions : MPMI·Helge WeingartMatthias S Ullrich
Aug 18, 2005·Environmental Microbiology·Maria L MarcoSteven E Lindow

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Citations

Apr 9, 2008·Applied and Environmental Microbiology·Savina O StoitsovaHelge Weingart
Jun 2, 2015·Frontiers in Microbiology·Gabriela Alfaro-Espinoza, Matthias S Ullrich
Apr 24, 2008·FEMS Microbiology Letters·Angela V SmirnovaMatthias S Ullrich
May 2, 2018·FEBS Letters·Tatsuya NoboriKenichi Tsuda
Apr 9, 2020·Extremophiles : Life Under Extreme Conditions·Jasmina VandrichHans Jörg Kunte

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